Reducing malaria transmission

Abstract

The present invention relates to compositions comprising bacteria of the genus Delftia, in particular compositions comprising Delftia tsuruhatensis, and the bacteria themselves, for use in preventing the transmission of malaria, and methods of reducing the transmission of malaria and / or Plasmodium using the compositions of the invention.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of malaria and provides compositions and methods useful for preventing the spread of malaria. BACKGROUND

[0002] Parasitic protozoan infections are responsible for a wide range of medically important diseases including malaria.

[0003] Malaria is a mosquito-borne disease that in humans can be caused by five Plasmodium parasites, of which P. falciparum is the most virulent.

[0004] There is an urgent need to develop and implement additional tools to block the transmission of malaria that can be integrated into existing approaches to achieve the goal of elimination. SUMMARY

[0005] According to a first aspect of the invention there is provided a composition comprising bacteria of the genus Delftia.

[0006] According to a second aspect of the invention there is provided a method of reducing or preventing the transmission of Plasmodium comprising the step of contacting one or more mosquitoes with bacteria of the genus Delftia.

[0007] In another aspect the invention provides bacteria of the genus Delftia for use in reducing the transmission of malaria.

[0008] In yet another aspect there is provided a mosquito of the genus Anopheles colonised with bacteria of the genus Delftia (hereinafter referred to as a mosquito of the invention).

[0009] In particular embodiments of these aspects the bacteria of the genus Delftia is Delftia tsuruhatensis.

[0010] The invention can be advantageous in many ways. The inventors have discovered that bacteria of the genus Delftia, in particular Delftia tsuruhatensis, hinders the transmission of Plasmodium parasites in mosquitoes. When introduced into an environment containing mosquitoes, the compositions of the invention prevent the transmission of Plasmodium by inhibiting the ookinete and oocysts of Plasmodium in the gut of mosquitoes. Bacteria of the genus Delftia, in particular D. tsuruhatensis, can be used as a tool against the spread of malaria. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Colony morphology of Delftia tsuruhatensis is shown.

[0012] Figure 2Show the effect of Delftia to Plasmodium falciparum oocyst intensity.Each point represents the sum of oocysts in the midgut of a single mosquito, and horizontal line illustrates average oocyst infection intensity.Mann Whitney test is used to compare the statistical significance between different treatments and the control (the processing that shows statistically significant distribution is represented by asterisk: p < 0.1, p < 0.01, p < 0.001, p < 0.0001 is represented by *, **, ***, **** respectively, and ns=is not significant). The table below the figure describes the sum, infection rate %, transmission blocking potential, average oocyst intensity reduction and average oocyst intensity of the well-fed mosquitoes that each process is dissected.Transmission blocking potential percentage and average oocyst intensity are reduced by using the value normalization from the control group. Figure 2 A and 2B represent the results of two independent experiments. DETAILED DESCRIPTION

[0013] In one aspect, the present invention provides a composition comprising bacteria of the genus Delftia. Delftia is a Gram-negative bacterium belonging to the class Betaproteobacteria and the family Comamonadaceae.

[0014] The bacterium of the genus Delftia can be any bacterium of the genus Delftia. In one embodiment of the present invention, the bacterium of the genus Delftia is Delftia. The bacterium was deposited on May 21, 2019, under the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland) under accession number NCIMB 43398. The bacterium was isolated and identified as Delftia by 16S rRNA sequencing. It is a Gram-negative bacterium belonging to the class Betaproteobacteria and the family Comamonadaceae. In one embodiment of the present invention, the bacterium of the genus Delftia is a bacterium deposited on May 21, 2019, under the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland) with accession number NCIMB 43398.

[0015] In one embodiment of the invention, the composition is suitable for reducing or preventing: (i) malaria and / or (ii) Plasmodium transmission in mosquitoes. In one embodiment, the composition is suitable for preventing malaria transmission in mosquitoes. In another embodiment, the composition is suitable for preventing Plasmodium transmission in mosquitoes.

[0016] As defined herein, "reducing or preventing malaria or Plasmodium transmission" is defined as stopping malaria, for example by inhibiting the mosquito stage (oocyst formation) of the Plasmodium.

[0017] It has been discovered that Delftia bacteria, particularly Delftia spp., can inhibit the transmission of malaria in mosquitoes by blocking the Plasmodium. In particular, it has been shown herein that when introduced into an environment containing mosquitoes, Delftia spp. can prevent the transmission of Plasmodium by inhibiting the kinetozoa and oocysts of Plasmodium species in the mosquito midgut. Thus, the compositions of the present invention can reduce or prevent the transmission of malaria and / or Plasmodium in mosquitoes.

[0018] The mosquito can be any mosquito capable of transmitting malaria, such as a mosquito of the genus Anopheles. It is contemplated that the compositions and methods of the present invention extend to any species of mosquito of the genus Anopheles. In one embodiment of the present invention, the mosquito is Anopheles gambiae or Anopheles stephensi. In one embodiment, the mosquito is Anopheles stephensi. In another embodiment, the mosquito is Anopheles gambiae.

[0019] The malarial parasite may be any malarial parasite. In one embodiment, the malarial parasite is a Plasmodium parasite. In one embodiment, the parasite is Plasmodium falciparum. In another embodiment, the parasite is Plasmodium berghei.

[0020] The compositions of the present invention may be in any suitable form and may include any suitable carrier.

[0021] The composition can be a feed composition, i.e., the composition can be in a form that is provided to mosquitoes for consumption. In one embodiment, the feed composition is a sugar source or a nectar feed. In one embodiment, the feed composition is a sugar source. The sugar source can be an attractive sugar bait, or be contained within an attractive sugar bait. An attractive sugar bait comprises sugar and a toxic component. An attractive sugar bait according to the present invention comprises the composition of the present invention without the toxic component, i.e., comprises sugar and the composition of the present invention.

[0022] In one embodiment, the composition is in the form of a bait. The bait is designed to attract mosquitoes to contact the composition. In one embodiment, upon contact with the mosquito, the composition is then internalized by the mosquito, for example, through ingestion. An attractant may also be used. The attractant may be a pheromone, such as a male or female pheromone. The attractant is used to attract the mosquito to the bait. The bait may be in any suitable form, such as a solid, paste, pellet, or powder.

[0023] The bait may be provided in a suitable "housing" or "trap". Such housings and traps are commercially available, and existing traps may be adapted to include the composition of the present invention. The housing or trap may, for example, be box-shaped and can be provided in a pre-formed state, or may, for example, be formed from foldable cardboard. Suitable materials for the housing or trap include plastics and cardboard, particularly corrugated cardboard. The inner surface of the trap may be lined with a sticky substance to restrict the movement of the mosquito once it is inside the trap. The housing or trap may contain suitable grooves in which the bait may be held in place. The trap is distinguished from the housing in that the mosquito cannot easily leave the trap after entering, whereas the housing acts as a "feeding station", which provides the mosquito with a preferred environment in which it can feed and feel safe from predators.

[0024] In a second aspect, the present application provides a method of reducing or preventing the transmission of a Plasmodium by a mosquito, the method comprising the step of contacting one or more mosquitoes with a bacterium of the genus Delphinius.

[0025] In one embodiment of the application, the bacterium of the genus Delphinius is Delphinius delphinius.

[0026] In another embodiment, the bacterium of the genus Delphinius is the bacterium deposited under accession number NCIMB 43398 on 21 May 2019 under the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland).

[0027] The step of contacting the mosquito with the bacterium can be carried out in any suitable way. For example, the person need not physically contact the mosquito and the bacterium of the genus Delphinius, they can leave the bacterium in a place where they know that the mosquito will come into contact with it.

[0028] The bacterium can be in the form of a composition of the application as described in the first aspect of the application.

[0029] In certain embodiments of the application, the contacting can be achieved by treating an area with a composition of the application, for example by using a spray formulation, such as an aerosol or pump spray. In certain embodiments of the application, the area can be treated via aerial delivery, for example by a vehicle mounted device or the like. In some embodiments, the spray composite is sprayed, for example by backpack spraying, aerial spraying, spray / dust or the like.

[0030] The mosquito can be any mosquito capable of transmitting malaria, for example a mosquito of the genus Anopheles. The compositions and methods of the application can extend to any Anopheles species of mosquito. In one embodiment of the application, the mosquito is Anopheles gambiae or Anopheles stephensi. In one embodiment, the mosquito is Anopheles stephensi. In another embodiment, the mosquito is Anopheles gambiae.

[0031] The Plasmodium can be any Plasmodium. In one embodiment, the Plasmodium is a Plasmodium parasite. In one embodiment, the parasite is Plasmodium falciparum. In another embodiment, the parasite is Plasmodium berghei.

[0032] In a third aspect, the present invention provides a Delftia bacterium for use in reducing the transmission of malaria. In one embodiment, the Delftia bacterium is Delftia. The bacterium may be in the form of a composition as described above in the first aspect of the invention. In one embodiment, the Delftia bacterium is used to reduce the transmission of malaria in mosquitoes. In this context, reducing the transmission of malaria may also be understood to mean "reducing the transmission of Plasmodium."

[0033] The mosquito can be any mosquito capable of transmitting malaria, such as a mosquito of the genus Anopheles. In one embodiment of the present invention, the mosquito is Anopheles gambiae or Anopheles stephensi. In one embodiment, the mosquito is Anopheles stephensi. In another embodiment, the mosquito is Anopheles gambiae.

[0034] The malarial parasite can be any malarial parasite. In one embodiment, the malarial parasite is a Plasmodium parasite. In one embodiment, the parasite is Plasmodium falciparum. In another embodiment, the parasite is Plasmodium berghei.

[0035] In another aspect of the invention, bacteria of the genus Delftia can be used in disease control strategies based on direct exposure of larval or adult mosquitoes to the bacteria. Exposure can be achieved through targeted administration of the bacteria or through interaction of natural mosquito populations with mosquitoes already colonized with the bacteria of the genus Delftia.

[0036] The mosquitoes of the present invention containing the Delftia bacteria can be used as agents that are able to mate with other mosquito populations in an area and thus spread the bacteria to other mosquito populations in an area. Mosquitoes containing the Delftia bacteria can be easily generated by infecting a suitable mosquito species with the desired Delftia strain.

[0037] Alternatively, the bacteria could be deployed directly to colonize local mosquito populations.

[0038] The Anopheles mosquito can be exposed to the Delftia bacteria from another mosquito, or directly to the bacteria themselves. If the Delftia bacteria are used directly (i.e., not via another mosquito), the bacteria can be delivered by any suitable method and combined with the delivery agent in any suitable manner that allows the composition to be administered to the mosquito. For example, the mosquito can be contacted with the bacteria in pure or substantially pure form (e.g., a solution containing Delftia).

[0039] In a specific embodiment, the Delftia bacterium is in a composition with a delivery agent.

[0040] In another specific embodiment, the mosquito larval forms can simply be "drenched" or "sprayed" with a solution containing the bacteria.

[0041] Alternatively, the composition comprising Delftia can be combined with a mosquito food component, such as artificial nectar or sugar bait, to facilitate delivery and / or thereby increase mosquito uptake of the composition. Methods for oral administration include, for example, directly mixing the composition with mosquito food, spraying the composition in mosquito habitats, or fields including areas of stagnant water. The composition can also be introduced into a culture medium in which mosquitoes grow, survive, reproduce, feed, or infest.

[0042] In another embodiment, the composition is in the form of a bait. The bait is designed to attract mosquitoes to contact the composition. In one embodiment, upon contact with the mosquito, the composition is then internalized by the mosquito, for example, through ingestion. The bait may depend on the target species. An attractant may also be used. The attractant may be a pheromone, such as a male or female pheromone. The attractant is used to attract the mosquito to the bait. The bait may be in any suitable form, such as a solid, paste, pellet, or powder.

[0043] The bait may be provided in a suitable "housing" or "trap". Such housings and traps are commercially available, and existing traps may be adapted to include the composition of the present invention. The housing or trap may, for example, be box-shaped and can be provided in a pre-formed state, or may, for example, be formed from foldable cardboard. Suitable materials for the housing or trap include plastics and cardboard, particularly corrugated cardboard. The inner surface of the trap may be lined with a sticky substance to restrict the movement of the mosquito once it is inside the trap. The housing or trap may contain suitable grooves in which the bait may be held in place. The trap is distinguished from the housing in that the mosquito cannot easily leave the trap after entering, whereas the housing acts as a "feeding station", which provides the mosquito with a preferred environment in which it can feed and feel safe from predators.

[0044] In certain embodiments of the present invention, an area can be treated with a composition of the present invention, for example, by using a spray formulation, such as an aerosol or pump spray. In certain embodiments of the present invention, an area can be treated via air delivery, for example, from a vehicle-mounted device, etc. In some embodiments, the composite material is sprayed, for example, by backpack spraying, airplane spraying, spray / dusting, etc.

[0045] The mosquitoes according to the invention are not able to transmit Plasmodium, thus making them suitable agents for preventing the transmission of malaria in addition to the direct use of Delftia bacteria.

[0046] The present invention can be deployed alongside other malaria eradication efforts. For example, the compositions, methods, and bacteria used in the present invention can be used in conjunction with known antimalarial agents. In one embodiment, the compositions or bacteria used in the present invention can be used in combination with one, two, or three additional antimalarial agents. Integrated Vector Management (IVM) recommends taking full advantage of available tools.

[0047] The at least one other antimalarial agent may also be selected from ferroquine, KAF156, sipagamine, DSM265, artemisinone, artefenomel, MMV048, SJ733, P218, MMV253, PA92, DDD498, AN13762, DSM421, UCT947, ACT 451840, OZ609, OZ277 and SAR97276.

[0048] In the treatment of Plasmodium falciparum infection, the at least one, two or three additional antimalarial agents may be selected from the following list, wherein at least one antimalarial agent is an artemisinin-based agent: artemether+lumefantrine, artesunate+amodiaquine, artesunate+mefloquine, dihydroartemisinin+piperaquine, or artesunate+sulfadoxine-pyrimethamine (SP).

[0049] The above combination therapies are called artemisinin-based combination therapies (ACTs). The selection of ACTs is usually based on the results of efficacy studies against local strains of Plasmodium falciparum malaria.

[0050] In the treatment of P. vivax infection, an ACT as described above may be used. Alternatively, the at least one other antimalarial agent may be chloroquine, particularly in areas where chloroquine-resistant P. vivax malaria is not present. In areas where resistant P. vivax malaria has been identified, the infection may be treated with an ACT as described above.

[0051] The combination of therapeutic agents may conveniently be presented for use in the form of a pharmaceutical composition or formulation and may be administered together or separately, and when administered separately this can occur separately or sequentially in any order (by the same or by different routes of administration).

[0052] The compositions or bacteria used in the present invention can be used in conjunction with insecticide-treated mosquito nets (ITNs), including long-lasting insecticidal nets (LLINs) and / or indoor residual sprays (IRS). ATSB (attractive toxic sugar bait) lures mosquitoes to feed on sugars containing toxic mosquito-killing compounds. For efficiency, ATSB can also include Delftia bacteria, or, as discussed above, use Delftia bacteria in place of toxic compounds.

[0053] The following non-limiting examples illustrate the invention.

[0054] Example

[0055] Example 1

[0056] Isolation and identification of microorganisms from a population of Anopheles stephensi mosquitoes following the observation that the population was incapable of infection by Plasmodium falciparum

[0057] It was observed that the mosquito population could not be infected by P. falciparum.

[0058] In an effort to establish factors that may influence the loss of susceptibility of GSK Anopheles stephensi mosquitoes to P. falciparum, the mosquito midgut tissue was studied, as well as the reproductive fitness, survival, and microbial flora present in different mosquito stages and the environment used to breed these mosquitoes.

[0059] The Anopheles stephensi mosquito colony was initially established in August 2012, with eggs imported from Imperial College, London. Female adult mosquitoes were routinely used for P. falciparum infection in the Standard Membrane Feeding Assay (SMFA), and the mosquitoes displayed good midgut oocyst density (2 to 25 oocysts per mosquito) and prevalence (80 to 100%). The colony gradually lost susceptibility to P. falciparum after one year of colonization. In this study, we explored several different factors, including the microbial communities present in different mosquito tissues and the different laboratory breeding environments, breeding conditions, food, temperature, water, and other factors that may contribute to this loss of susceptibility.

[0060] method

[0061] Mosquito survival and reproduction

[0062] Adult female mosquito mortality was monitored. The percentage of mosquitoes that fed with mouse blood (during the breeding period) and human blood (during the SMFA period) was recorded. The number of eggs laid after feeding with mouse blood was also recorded.

[0063] Sample collection and analysis from incubators (surfaces, humidifiers, and water) used to breed mosquitoes Sample type: Swabs from the inner wall of the incubator (1-6)

[0064] Sample source: The established swab collection area comes from: the inner bottom plate, the inner top plate, and the two inner side walls

[0065] Sampling method: The established areas (described above) were swabbed with a cotton swab at the base. Each swab from the corresponding area was placed in 5 mL of sterile LB medium in a test tube. Each test tube was labeled with the date of collection, incubator number, and sampling location within the incubator.

[0066] These were then incubated at 37°C on a rotary shaker and screened for the presence of turbidity at regular time intervals; O / N, 24 hours, 48 ​​hours, 96 hours.

[0067] Sample Type: Water humidifier source and water sink in incubators (Incubators 5 & 6) used for propagation purposes

[0068] Sample source: Water from the humidifier container (in the event that the water container was empty, a swab was obtained from inside the receiver and processed as described above).

[0069] Sampling Method: Collect a 250 ml water sample from a water receiver into a sterile glass bottle. Then, filter it through a 0.22 micron filter placed in a vacuum filtration apparatus. Remove the filter with tweezers and place it in a test tube containing 5 mL of sterile LB medium. Gently rinse the filter with LB. Each test tube is labeled with the date of collection, incubator number, and sampling location within the incubator.

[0070] These were then incubated at 37°C on a rotary shaker and screened for the presence of turbidity at regular time intervals; O / N, 24 hours, 48 ​​hours, 96 hours.

[0071] Sample analysis

[0072] The results from above were tabulated in an Xcel spreadsheet.

[0073] A selected set of samples was sent to an external analytical group (DYNAMIMED SL) for bacterial identification analysis.Bacterial colonies were identified by biochemical characterization using standard biochemical tests.

[0074] Sample collection of mosquito tissues (eggs, larvae, whole mosquitoes, midgut)

[0075] Sample sources: Mosquito tissues from different developmental stages were freshly collected in sterile eppendorfs with 100 μl sterile PBS and immediately stored at -80°C for further use. Tissues included: eggs (collected from moistened filter paper); whole larvae from different instars (10-12 / vial); midgut tissues of adult female mosquitoes were microdissected, and a total of 10-12 midguts were used; and whole adult female mosquitoes (10-12 / vial) - no PBS was added, but they were directly stored in sterile eppendorfs at -80°C.

[0076] Analysis of samples from mosquito tissue

[0077] Mosquito tissue samples were homogenized using a KONTES handheld automatic homogenizer equipped with a sterile disposable pestle. The entire homogenate was transferred to a sterile LB broth or YESP broth in a glass test tube (5-10 mL) and incubated at 27° C. on a rotary shaker. When turbidity was observed, the sample was plated on solid agar (LB or YESP) to obtain a single, purer, different bacterial colony. Single individual bacterial colonies were selected based on different colony morphologies and transferred to fresh liquid culture medium. Overnight grown cultures (500 μl) were thoroughly mixed with an equal volume of 80% sterile glycerol and stored frozen at -80° C.

[0078] Sample analysis

[0079] Bacterial samples isolated from the different tissues mentioned above were sent to an external analysis group (DYNAMIMED SL) for identification.Bacterial colonies were identified by biochemical characterization using standard biochemical tests.

[0080] Microscopic observation of midgut tissue

[0081] The midguts of mosquitoes (adult females) of different ages after eclosion were dissected (Leica, M80) and mounted on the glass side in sterile PBS or incubated in D / W containing 0.2% mercurochrome solution for 10-15 minutes. Individual midguts were observed using a light microscope (Leica, DM2000) with a 10X or 40X objective (100X or 400X total magnification) or at 100X using oil immersion by placing a drop of oil on a glass coverslip.

[0082] result

[0083] Mosquito survival and reproduction

[0084] The Anopheles stephensi CRESA colony was established at the Tres Cantos facility in August 2012, with eggs imported from CRESA, Barcelona. The mosquito colony was initially established at CRESA, with eggs obtained from Imperial College London (Robert Sinden Group). The mosquito survival, reproductive capacity, and overall quality of the mosquitoes were not limited over time. No abnormal mortality rates were observed. The percentage of mosquitoes fed on human blood was no less than 60% to 70%, the average feeding rate observed in mosquitoes from this colony.

[0085] Table 1: Results of bacterial identification by biochemical characterization

[0086]

[0087]

[0088]

[0089] All these samples showed the dominance of one major bacterium, which was initially identified as Pseudomonas oryzae by biochemical characterization (API strips from Biomerieux). These samples were reanalyzed by 16S rRNA typing, and two different bacteria were identified: Delftia sp. Figure 1 ) and Pseudomonas putida. The dominant bacterium was identified as Delftia by 16S rRNA typing. Another bacterium, Pseudomonas putida, was also isolated from mosquito tissues, but it was not as dominant as Delftia.

[0090] Example 2

[0091] Delftia and malaria transmission: proof of concept experiments establishing the role of Delftia in interrupting Plasmodium falciparum transmission

[0092] To evaluate the ability of Delftia to inhibit Plasmodium falciparum infection in Anopheles stephensi mosquitoes.

[0093] The Anopheles stephensi mosquito colony was initially established in August 2012 at the GSK, Tres Cantos, Spain facility, with eggs imported from Imperial College, London. Female adult mosquitoes were routinely used for P. falciparum infection in the Standard Membrane Feeding Assay (SMFA), and the mosquitoes showed good average midgut oocyst density (2 to 25 oocysts per mosquito) and prevalence (80 to 100%). The colony gradually lost its susceptibility to P. falciparum after one year of colonization. Although the colony's fitness and reproductive capacity were not compromised, we observed that the mosquito midgut showed the appearance of refractive microscopic crystal-like structures dispersed throughout the membrane and no oocysts were present. Mosquito tissues were screened for the presence of both fungi and bacteria, and we confirmed the presence of at least one bacterial species that was predominant in all tissues. We explored several different factors, including breeding conditions, food, temperature, and water, that could contribute to the loss of susceptibility. One factor that may have contributed to the abundance of this particular bacterial type may be related to the breeding protocol used at the time. Due to a lack of susceptibility to Plasmodium spp., the mosquito colony ceased. Mosquito tissue samples (larvae, male and female adults, and midgut tissue) were stored at -80°C. By 16S rRNA typing, the dominant bacterium was identified as Delftia spp. Another bacterium, Pseudomonas putida, was also isolated from mosquito tissue, but it was less dominant than Delftia spp.

[0094] Processing of cryopreserved mosquito tissue samples

[0095] Cryopreserved tissue samples of Anopheles stephensi larvae and adult female midguts were selected from GSK glycerol stocks grown in LB broth at 27°C and stored at -80°C. These samples were obtained from a colony of Anopheles stephensi established in 2012 that is incapable of transmitting Plasmodium falciparum. Three different vials were selected and labeled as numbers 5, 11, and 28;

[0096] n° 5 = midgut from 5-8 day old mosquitoes.

[0097] n° 11 = midgut obtained from 26-30 day old adult worms.

[0098] n°28 = Large colony isolated from homogenized stage II larvae.

[0099] To isolate individual colonies from each sample, a sterile loop was introduced into each tube and inoculated into 20 ml of LB broth (Delftia spp., which can grow at both 37°C and 27°C) maintained at 200 rpm overnight at 37°C. The samples were streaked on LB plates and kept at 37°C for 24-48 hours. Pure colonies were sent to an external laboratory (DYNAMIME D.L.) for identification. All these samples showed a predominance of one major bacterial species, which was initially identified as Pseudomonas oryzae by biochemical characterization (API strips from Biomerieux). However, these samples were subsequently reanalyzed by 16S rRNA typing, and two different bacteria were identified: Delftia ( Figure 1 ) and other Pseudomonas putida spp. Figure 1 The colony morphology of Delftia spp. is shown: creamy white round colonies, Gram-negative, rod-shaped, catalase- and oxidase-positive, motile bacteria from the family Comamonadaceae.

[0100] Mosquito rearing and antibiotic treatment for removal of bacterial flora from the midgut

[0101] Cyclic colonies of Anopheles stephensi were maintained in a controlled climate chamber (Panasonic MLR352 PE) at 26.5 ± 1°C, a 14 L:10 D photoperiod, and 75 ± 5% RH. ) received ad libitum access to 10% sucrose ( S7903) / aqueous solution + 1% The larvae were reared in plastic trays (20 x 15 x 6 cm) in groups of 250 larvae per tray and hydrated with powdered Goldfish stick feeding.

[0102] To remove the normal mosquito midgut flora, the cells were washed with 1X penicillin-streptomycin [ P4333] and 0.4% gentamicin [ Newly emerged mosquitoes were fed a 10% sucrose solution in sterile distilled water containing [G1397]. The cotton was replaced with fresh antibiotic solution every 48 hours. Two days before the experiment, female mosquitoes were gently transferred to a cardboard container sealed with a double layer of mesh. These mosquitoes were starved overnight by deprivation of sugar-soaked cotton. To determine the effectiveness of the antibiotic treatment, midguts from 10-12 mosquitoes were dissected, homogenized in PBS, and plated on LB agar.

[0103] Bacterial sample preparation and recolonization of the Anopheles stephensi midgut

[0104] Using pure colonies of Delftia from LB plates, bacterial samples for introduction into mosquitoes were prepared (see Figure 1 A single bacterial colony was grown in 100 ml LB broth at 37 °C and 200 rpm overnight to an OD of 1.0. 600 {10 6 8 ml of culture were pelleted (centrifugation for 2 min at 10,000 g), washed twice in 10% sucrose solution, and finally resuspended in 4 ml of 10% sucrose solution (final OD 600 The suspension was introduced into a small (5 mL) glass test tube covered with a cotton plug. The tube was flipped so that the cotton completely absorbed the suspension and then introduced into a mosquito cup to allow feeding. On the day of the experiment, mosquitoes were allowed to feed on cotton soaked with 10% sugar solution + bacteria for 2 hours. Antibiotic-treated mosquitoes (40 to 50 female adults / cup as described above) were starved for 24 hours and then fed with the bacterial suspension. Subsequently, all cotton was replaced with fresh cotton soaked in 10% sugar solution. Untreated mosquitoes (control) were provided with a 10% sucrose solution without bacterial suspension.

[0105] Counting mosquito midgut bacteria load

[0106] Under a stereomicroscope, mosquitoes were carefully dissected on slides containing sterile PBS. The midguts of 10 female mosquitoes (in each treatment) were quickly introduced into 100 μL sterile PBS in 1.5 mL eppendorf tubes. The samples were homogenized using a handheld homogenizer and serial dilutions were performed. 100 μL of each dilution was plated onto LB agar plates and incubated at 37°C for 24-48 hours. Bacterial colonies were counted and CFU / ml was determined.

[0107] The role of Delftia in the transmission of Plasmodium falciparum

[0108] To determine the role of Delftia in Plasmodium transmission, bacterially colonized mosquitoes (as described above) were fed with mature gametocytes in a SMFA as follows. Table 1 describes the different treatments in the control and test groups.

[0109] Table 1: Experimental treatments designed to determine the establishment of Delftia in the female mosquito midgut (treatments 1 & 2) and the role of Delftia in Plasmodium transmission (treatment 3).

[0110]

[0111] Gametocyte production

[0112] Gametocyte cultures were generated using a strain producing P. falciparum NF54 gametocytes kindly provided by M. Delves (Imperial College, London). Asexual propagation was maintained at a maximum total parasitemia of 1% in RPMI medium supplemented with 10% human serum. The gametocyte culture protocol was adapted from that described by Ifediba and Vanderberg (1981). Gametocyte induction was initiated at a parasitemia of 0.5% (>70% parasitemia) and a hematocrit of 4% in RPMI medium supplemented with 5% human serum A+ and 0.5% Albumax (gametocyte production medium RPMI 1640 (Sigma R5886), 1500 ml, 30 mM bicarbonate (Sigma-Aldrich S5761), 5 mM hypoxanthine (Gibco 11067-030), supplemented with 5% human serum A+ and 0.5% Albumax).

[0113] The culture was maintained for up to 20 days with daily culture medium changes without the addition of fresh RBCs. On days 13 to 15 after induction, the culture medium was replaced with gametocyte-only serum medium {RPMI1640 (15.87 g) containing L-glutamine and 25 mM HEPES (Gibco 13018-031), 10 mM glucose (Sigma-Aldrich G8270); 20 mM bicarbonate (Sigma-Aldrich S5761); 5 mM hypoxanthine (Gibco 11067) / L, culture medium supplemented with 10% human A+ serum (Interstate Blood Bank)}. After 13 days, the culture was monitored daily for phase V gametocyte chromatinization, male and female gametocyte ratios using Giemsa-stained smears, and viability was monitored by performing a microgametogenesis assay.

[0114] Standard Membrane Feeding Assay (SMFA)

[0115] Two days before the start of the SMFA, antibiotic-treated mosquitoes were fed with the Delftia species described above. On the day of feeding, mature gametocyte cultures were centrifuged at 2500 × g for 3 minutes at 37°C. The supernatant was removed, and the pellet was diluted 1:1 with fresh human RBCs of 100% hematocrit, and finally an artificial mosquito blood meal was prepared with pre-warmed human serum at 50% hematocrit. All steps were performed at 37°C. A blood meal that did not contain gametocytes was prepared in a similar manner. The prepared blood meal was fed to female Anopheles stephensi mosquitoes in duplicate via Parafilm membranes for a duration of 30-40 minutes, which were attached to a glass feeder (Fisher Scientific, #12831283) connected to a 37°C circulating water bath. The fed mosquitoes were maintained in an incubator at 26.5 ± 1°C, 14L:10D photoperiod, and 75 ± 5% RH. 24 hours after blood feeding, the midguts of mosquitoes were carefully dissected and the bacterial count was determined as described above. To count midgut oocysts, the midguts of mosquitoes with fully developed ovaries (fed mosquitoes) were dissected 7-8 days after feeding (Leica, M80) and incubated in D / W containing 0.2% mercurochrome solution for 10-15 minutes. Using an optical microscope (Leica, DM2000), the total number of oocysts in each midgut was counted using a 10X objective lens (100X magnification). Both the infection rate (the percentage of mosquitoes with one or more oocysts) and the average oocyst infection intensity were defined in each treatment.

[0116] result

[0117] Delftia can successfully establish in the midgut of Anopheles stephensi

[0118] To determine whether Delftia species effectively colonizes the mosquito midgut, mosquitoes fed with Delftia were dissected at two different time points: before an infectious blood (SMFA) feeding and 24 hours after the blood feeding. Midguts from 10 mosquitoes were pooled, homogenized in 100 μL of PBS, plated on LB agar, and colony forming units (CFU) was determined. Only one type of colony was observed. The colony morphology and characteristics of the recovered bacteria (although in very low numbers) were identical to those of Delftia species, confirming that Delftia species can colonize the mosquito midgut (Table 2). Bacterial numbers increased by two to three orders of magnitude after the blood feeding (consistent with reports and studies showing an increase in midgut microbiota after blood feeding). Midguts from mosquitoes from the untreated control group showed no colonies before or after the blood feeding, demonstrating the effectiveness of the gentamicin-penicillin-streptomycin treatment in removing the resident microbiota (Table 2).

[0119] Table 2: Delftia species bacteria recovered from the midgut of female Anopheles stephensi before and after blood feeding. The table shows the log CFU / ml in the control and test groups.

[0120]

[0121] Delftia significantly reduced the mean oocyst density and infection rate of Plasmodium falciparum in Anopheles stephensi mosquitoes

[0122] A greater than 70% reduction in mean oocyst density was observed in recolonized mosquitoes compared to untreated controls. The results showed that the transmission blockade of treated mosquitoes was greater than 60% ( Figure 2 ).

[0123] The gentamicin-penicillin-streptomycin treatment employed successfully removed the inherent culturable bacterial midgut flora present in GSK Anopheles stephensi mosquitoes. Delftia bacterial colonies were successfully detected in the midguts of mosquitoes from either sugar-fed or blood-fed mosquitoes. 24 hours after the blood meal, blood-fed mosquitoes showed a two- to three-fold increase in bacterial numbers compared to sugar-fed mosquitoes. A reduction of more than 70% in the mean oocyst intensity of Plasmodium falciparum and a 60% transmission blockade were detected compared to untreated controls. Invasion of the mosquito midgut by Plasmodium falciparum kinetozoa occurred between 16 and 24 hours after blood feeding. During this time period (24 hours after blood feeding), only Delftia bacterial colonies were recovered from the mosquito midgut, indicating that Delftia is directly involved in reducing the number of Plasmodium falciparum oocysts.

Claims

1. Use of a composition comprising Delftia tsuruhatensis with accession number NCIMB 43398 for reducing or preventing the transmission of Plasmodium in mosquitoes, wherein the Plasmodium is Plasmodium falciparum.

2. The use according to claim 1, wherein the mosquito is a mosquito of the genus Anopheles.

3. The use according to claim 2, wherein the mosquito is Anopheles gambiae or Anopheles stephensi.

4. The use according to claim 3, wherein the mosquito is Anopheles stephensi.

5. Use according to any one of the preceding claims, wherein the composition is in the form of a sugar source or nectar feed.

6. Use of a composition comprising Delftia tsuruhatensis with accession number NCIMB 43398 in the preparation of a medicament for reducing or preventing the transmission of Plasmodium in mosquitoes, wherein the Plasmodium is Plasmodium falciparum.

7. A method of reducing or preventing the transmission of Plasmodium, comprising the step of contacting one or more mosquitoes with Delftia tsuruhatensis, accession number NCIMB 43398, wherein the Plasmodium is Plasmodium falciparum.

8. The method of claim 7, wherein the mosquito is of the genus Anopheles.

9. The method of claim 8, wherein the mosquito is Anopheles gambiae or Anopheles stephensi.

10. The method of claim 9, wherein the mosquito is Anopheles stephensi.

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